CD20 and CD20 isoforms (“CD20”) are generally appreciated to be transmembrane proteins of the tetra-spanin family that are expressed on the surface of B-cells (Valentine et al., J. Biol. Chem., 264(19):11282-11287 (1989); Einfeld et al., EMBO J., 7(3):711-717 (1988)). CD20 is expressed by the vast majority of peripheral blood B-cells as well as B cells from various lymphoid tissues. CD20 expression generally persists from the early pre-B cell stage of development until the plasma cell differentiation stage of development (Tedder et al., J. Immunol., 135(2):973-979 (1985)). CD20 is not generally expressed by hematopoietic stem cells, pro-B cells, differentiated plasma cells or non-lymphoid tissues, or the like. In addition to expression in normal B-cells, CD20 is expressed in B-cell derived malignancies such as non-Hodgkin's lymphoma (NHL) and B-cell chronic lymphocytic leukemia (CLL) (Anderson et al., Blood, 63(6): 1424-1433 (1984)) and B cells involved in immune disorders, autoimmune disease and inflammatory diseases.
Although the exact function of CD20 is unclear, CD20 is implicated in calcium mobilization and may function as a calcium channel (Tedder et al., J. Cell Biochem., 14D:195 (1990)). CD20 might be involved in the activation and differentiation of B-cells (Tedder et al., Eur. J. Immunol., 16(8):881-887 (1986)).
The expression profile of CD20 and knowledge of existing CD20 antibodies has made CD20 a target of interest for antibody therapies. It is known in the art that, generally, antibodies for CD20 are classified based on their functional properties. For example, Type I antibodies are characterized by their ability to distribute CD20 into lipid raft compartments and, when chimerized, can mediate complement-dependent cytotoxicity (“CDC”) (Deans J P, J Biol Chem 1998; 273: 344-8; Cragg M S, Blood 2003; 101:1045-52; and Cragg M S Blood. 2004; 103:2738-2743). Type I antibodies typically do not have effective pro-apoptotic activity on their own, unless the antibodies are cross-linked. Examples of Type I antibodies requiring cross-linking are rituximab and 2F2 (Cragg et al., Blood, 101(3):1045-1052 (2003); and Teeling et al., Blood, 104(6):1793-1800 (2004)). In contrast, Type II antibodies are unable to distribute CD20 into lipid rafts. If chimerized, Type II antibodies have limited CDC activity. Type II antibodies are characterized by their strong pro-apoptotic activity. B1 and GA101 are examples of Type II antibodies (Cragg M S, Blood 2003; 101:1045-52; Cragg M S Blood. 2004; 103:2738-2743); and Umana et al., Blood, 108:72a, Abstract 229 (2006)). Type I and Type II antibodies can potentially mediate antibody-dependent cell-mediated cytotoxicity (ADCC).
Given the expression of CD20 by unwanted cells, such as in B-cell lymphomas, this antigen is useful for targeting harmful CD20 positive cells (e.g., lymphoma cells). In essence, such targeting is generalized as follows: antibodies specific to CD20 surface antigen of B cells are administered to a patient. These anti-CD20 antibodies specifically bind to the CD20 antigen of both normal and unwanted (e.g., malignant and immunoreactive) B cells; the antibody bound to the CD20 surface antigen may lead to the destruction and depletion of the B cells.
Additionally, chemical agents or radioactive labels having the potential to destroy CD20 expressing tumor cells can be conjugated to the anti-CD20 antibody such that the agent is specifically “delivered” to the neoplastic B cells. Irrespective of the approach, a primary goal is to destroy the unwanted cells; the specific approach can be determined by the particular anti-CD20 antibody that is utilized, and thus, the available approaches to targeting the CD20 antigen can vary considerably. The rituximab (RITUXAN®) antibody is a genetically engineered chimeric murine/human monoclonal antibody directed against CD20. Rituximab is the antibody called “C2B8” in U.S. Pat. No. 5,736,137 (Anderson et al.). Rituximab is currently approved for the treatment of relapsed or refractory follicular lymphoma (Leget et al., Curr. Opin. Oncol., 10:548-551 (1998)). Reports indicate that with weekly infusions, rituximab resulted in overall response rates of 48%. However, many patients do not respond to rituximab treatment and responding patients taking rituximab eventually relapse and often develop resistance to rituximab treatment. Relapse and resistance, for example, to currently available therapies necessitate discovery of new CD20 directed agents and therapies.
Because of the limitations of available antibodies, there are next generation antibody therapeutics for CD20 in development which aim to improve specific functional aspects of rituximab. For example, afutuzumab or GA 101 has been reported to have marginal improvement of pro-apoptotic activity over rituximab in vitro, but GA101 fails to demonstrate CDC activity (Umana P, Blood 2006; 108:72a, Abstract 229 and WO 2005/044859). In addition, GA101 is a glycoengineered humanized antibody with improved ADCC activity (Umana et al., Blood 2006; 108:72a, Abstract 229; and WO 2005/044859). In the case of ofatumumab, a human monoclonal 2F2 antibody, improved in vitro CDC activity has been reported, especially in cells having lower CD20 antigen density (Teeling et al., J. Immunol., 177(1):362-371 (2006)). It has been proposed that this improved CDC activity results from the ability of 2F2 and related antibodies to recognize a novel epitope on CD20. Although ofatumumab with improved CDC activity is effective and was approved for treating patients with refractory chronic lymphocytic leukemia, ofatumumab was not active on rituximab-refractory NHL patients. American Society of Hematology Meeting 2009, Abstract #935.
Other compounds, in varying stages of development, have been reported to marginally improve certain intrinsic properties of rituximab or other known anti-CD20 antibodies. However, until now, no novel molecule having unique physical and functional features addressing the problems known in the art was available.
Rituximab has also been approved in the United States in combination with MTX to reduce signs and symptoms in adult patients with moderately- to severely-active RA who have had an inadequate response to at least one TNF antagonist. Many studies address the use of rituximab in a variety of non-malignant autoimmune or inflammatory disorders, including RA, in which B cells and autoantibodies appear to play a role in disease pathophysiology. Edwards et al., Biochem Soc. Trans. 30:824-828 (2002). Targeting of CD20 using anti-CD20 antibody has been reported to potentially relieve signs and symptoms of, for example, RA (Leandro et al., Ann. Rheum. Dis. 61:883-888 (2002); Edwards et al., Arthritis Rheum., 46 (Suppl. 9): S46 (2002); Stahl et al., Ann. Rheum. Dis., 62 (Suppl. 1): OP004 (2003); Emery et al., Arthritis Rheum. 48(9): 5439 (2003)), lupus (Eisenberg, Arthritis. Res. Ther. 5:157-159 (2003); Leandro et al. Arthritis Rheum. 46: 2673-2677 (2002); Gorman et al., Lupus, 13: 312-316 (2004)), immune thrombocytopenic purpura (D'Arena et al., Leuk. Lymphoma 44:561-562 (2003); Stasi et al., Blood, 98: 952-957 (2001); Saleh et al., Semin. Oncol., 27 (Supp 12):99-103 (2000); Zaja et al., Haematologica, 87:189-195 (2002); Ratanatharathorn et al., Ann. Int. Med., 133:275-279 (2000)), pure red cell aplasia (Auner et al., Br. J. Haematol., 116:725-728 (2002)); autoimmune anemia (Zaja et al., supra (erratum appears in Haematologica 87:336 (2002)), cold agglutinin disease (Layios et al., Leukemia, 15:187-8 (2001); Berentsen et al., Blood, 103: 2925-2928 (2004); Berentsen et al., Br. J. Haematol., 115:79-83 (2001); Bauduer, Br. J. Haematol., 112:1083-1090 (2001); Zaja et al., Br. J. Haematol., 115:232-233 (2001)), type B syndrome of severe insulin resistance (Coll et al., N. Engl. J. Med., 350:310-311 (2004), mixed cryoglobulinermia (DeVita et al., Arthritis Rheum. 46 Suppl. 9:S206/S469 (2002)), myasthenia gravis (Zaja et al., Neurology, 55:1062-1063 (2000); Wylam et al., J. Pediatr., 143:674-677 (2003)), Wegener's granulomatosis (Specks et al., Arthritis & Rheumatism 44:2836-2840 (2001)), refractory pemphigus vulgaris (Dupuy et al., Arch Dermatol., 140:91-96 (2004)), dermatomyositis (Levine, Arthritis Rheum., 46 (Suppl. 9):S1299 (2002)), Sjogren's syndrome (Somer et al., Arthritis & Rheumatism, 49:394-398 (2003)), active type-II mixed cryoglobulinemia (Zaja et al., Blood, 101:3827-3834 (2003)), pemphigus vulgaris (Dupay et al., Arch. Dermatol., 140:91-95 (2004)), autoimmune neuropathy (Pestronk et al., J. Neurol. Neurosurg. Psychiatry 74:485-489 (2003)), paraneoplastic opsoclonus-myoclonus syndrome (Pranzatelli et al. Neurology 60 (Suppl. 1) PO5.128:A395 (2003)), and relapsing-remitting multiple sclerosis (RRMS). Cross et al. (abstract) “Preliminary Results from a Phase II Trial of Rituximab in MS” Eighth Annual Meeting of the Americas Committees for Research and Treatment in Multiple Sclerosis, 20-21 (2003).
Patents and patent publications concerning CD20 antibodies, CD20-binding molecules, and self-antigen vaccines include U.S. Pat. Nos. 5,776,456, 5,736,137, 5,843,439, 6,399,061, and 6,682,734, as well as US 2002/0197255, US 2003/0021781, US 2003/0082172, US 2003/0095963, US 2003/0147885, US 2005/0186205, and WO 1994/11026 (Anderson et al.); U.S. Pat. No. 6,455,043, US 2003/0026804, US 2003/0206903, and WO 2000/09160 (Grillo-Lopez, A.); WO 2000/27428 (Grillo-Lopez and White); US 2004/0213784 and WO 2000/27433 (Grillo-Lopez and Leonard); WO 2000/44788 (Braslawsky et al.); WO 2001/10462 (Rastetter, W.); WO 2001/10461 (Rastetter and White); WO 2001/10460 (White and Grillo-Lopez); US 2001/0018041, US 2003/0180292, US 2002/0028178, WO 2001/34194, and WO 2002/22212 (Hanna and Hariharan); US 2002/0006404 and WO 2002/04021 (Hanna and Hariharan); US 2002/0012665, US 2005/0180975, WO 2001/74388, and U.S. Pat. No. 6,896,885B5 (Hanna, N.); US 2002/0058029 (Hanna, N.); US 2003/0103971 (Hariharan and Hanna); US 2005/0123540 (Hanna et al.); US 2002/0009444 and WO 2001/80884 (Grillo-Lopez, A.); WO 2001/97858; US 2005/0112060, US 2002/0039557, and U.S. Pat. No. 6,846,476 (White, C.); US 2002/0128448 and WO 2002/34790 (Reff, M.); WO 2002/060955 (Braslawsky et al.); WO 2002/096948 (Braslawsky et al.); WO 2002/079255 (Reff and Davies); U.S. Pat. Nos. 6,171,586 and 6,991,790, and WO 1998/56418 (Lam et al.); US 2004/0191256 and WO 1998/58964 (Raju, S.); WO 1999/22764 (Raju, S.); WO 1999/51642, U.S. Pat. No. 6,194,551, U.S. Pat. No. 6,242,195, U.S. Pat. No. 6,528,624 and U.S. Pat. No. 6,538,124 (Idusogie et al.); U.S. Pat. No. 7,122,637, US 2005/0118174, US 2005/0233382, US 2006/0194291, US 2006/0194290, US 2006/0194957, and WO 2000/42072 (Presta, L.); WO 2000/67796 (Curd et al.); WO 2001/03734 (Grillo-Lopez et al.); US 2002/0004587, US 2006/0025576, and WO 2001/77342 (Miller and Presta); US 2002/0197256 and WO 2002/078766 (Grewal, I.); US 2003/0157108 and WO 2003/035835 (Presta, L.); U.S. Pat. Nos. 5,648,267, 5,733,779, 6,017,733, and 6,159,730, and WO 1994/11523 (Reff et al.); U.S. Pat. Nos. 6,565,827, 6,090,365, 6,287,537, 6,015,542, 5,843,398, and 5,595,721 (Kaminski et al.); U.S. Pat. Nos. 5,500,362, 5,677,180, 5,721,108, 6,120,767, 6,652,852, and 6,893,625 as well as WO 1988/04936 (Robinson et al.); U.S. Pat. No. 6,410,391 (Zelsacher); U.S. Pat. No. 6,224,866 and WO00/20864 (Barbera-Guillem, E.); WO 2001/13945 (Barbera-Guillem, E.); WO 2000/67795 (Goldenberg); U.S. Pat. No. 7,074,403 (Goldenberg and Hansen); U.S. Pat. No. 7,151,164 (Hansen et al.); US 2003/0133930; WO 2000/74718 and US 2005/0191300A1 (Goldenberg and Hansen); US 2003/0219433 and WO 2003/68821 (Hansen et al.); WO 2004/058298 (Goldenberg and Hansen); WO 2000/76542 (Golay et al.); WO 2001/72333 (Wolin and Rosenblatt); U.S. Pat. No. 6,368,596 (Ghetie et al.); U.S. Pat. No. 6,306,393 and US 2002/0041847 (Goldenberg, D.); US 2003/0026801 (Weiner and Hartmann); WO 2002/102312 (Engleman, E.); US 2003/0068664 (Albitar et al.); WO 2003/002607 (Leung, S.); WO 2003/049694, US 2002/0009427, and US 2003/0185796 (Wolin et al.); WO 2003/061694 (Sing and Siegall); US 2003/0219818 (Bohen et al.); US 2003/0219433 and WO 2003/068821 (Hansen et al.); US 2003/0219818 (Bohen et al.); US 2002/0136719 (Shenoy et al.); WO 2004/032828 and US 2005/0180972 (Wahl et al.); and WO 2002/56910 (Hayden-Ledbetter). See also U.S. Pat. No. 5,849,898 and EP 330,191 (Seed et al.); EP332,865A2 (Meyer and Weiss); U.S. Pat. No. 4,861,579 (Meyer et al.); US 2001/0056066 (Bugelski et al.); WO 1995/03770 (Bhat et al.); US 2003/0219433 A1 (Hansen et al.); WO 2004/035607 and US 2004/167319 (Teeling et al.); WO 2005/103081 (Teeling et al.); US 2006/0034835, US 2006/0024300, and WO 2004/056312 (Lowman et al.); US 2004/0093621 (Shitara et al.); WO 2004/103404 (Watkins et al.); WO 2005/000901 (Tedder et al.); US 2005/0025764 (Watkins et al.); US 2006/0251652 (Watkins et al.); WO 2005/016969 (Carr et al.); US 2005/0069545 (Carr et al.); WO 2005/014618 (Chang et al.); US 2005/0079174 (Barbera-Guillem and Nelson); US 2005/0106108 (Leung and Hansen); US 2005/0123546 (Umana et al.); US 2004/0072290 (Umana et al.); US 2003/0175884 (Umana et al.); and WO 2005/044859 (Umana et al.); WO 2005/070963 (Allan et al.); US 2005/0186216 (Ledbetter and Hayden-Ledbetter); US 2005/0202534 (Hayden-Ledbetter and Ledbetter); US 2005/136049 (Ledbetter et al.); US 2003/118592 (Ledbetter et al.); US 2003/133939 (Ledbetter and Hayden-Ledbetter); US 2005/0202012 (Ledbetter and Hayden-Ledbetter); US 2005/0175614 (Ledbetter and Hayden-Ledbetter); US 2005/0180970 (Ledbetter and Hayden-Ledbetter); US 2005/0202028 (Hayden-Ledbetter and Ledbetter); US 2005/0202023 (Hayden-Ledbetter and Ledbetter); WO 2005/017148 (Ledbetter et al.); WO 2005/037989 (Ledbetter et al.); U.S. Pat. No. 6,183,744 (Goldenberg); U.S. Pat. No. 6,897,044 (Braslawski et al.); WO 2006/005477 (Krause et al.); US 2006/0029543 (Krause et al.); US 2006/0018900 (McLCCormick et al.); US 2006/0051349 (Goldenberg and Hansen); WO 2006/042240 (Iyer and Dunussi-Joannopoulos); US 2006/0121032 (Dahiyat et al.); WO 2006/064121 (Teillaud et al.); US 2006/0153838 (Watkins), CN 1718587 (Chen et al.); WO 2006/084264 (Adams et al.); US 2006/0188495 (Barron et al.); US 2004/0202658 and WO 2004/091657 (Benynes, K.); US 2005/0095243, US 2005/0163775, WO 2005/00351, and WO 2006/068867 (Chan, A.); US 2006/0135430 and WO 2005/005462 (Chan et al.); US 2005/0032130 and WO 2005/017529 (Beresini et al.); US 2005/0053602 and WO 2005/023302 (Brunetta, P.); US 2006/0179501 and WO 2004/060052 (Chan et al.); WO 2004/060053 (Chan et al.); US 2005/0186206 and WO 2005/060999 (Brunetta, P.); US 2005/0191297 and WO 2005/061542 (Brunetta, P.); US 2006/0002930 and WO 2005/115453 (Brunetta et al.); US 2006/0099662 and WO 2005/108989 (Chuntharapai et al.); CN 1420129A (Zhongxin Guojian Pharmaceutical); US 2005/0276803 and WO 2005/113003 (Chan et al.); US 2005/0271658 and WO 2005/117972 (Brunetta et al.); US 2005/0255527 and WO 2005/11428 (Yang, J.); US 2006/0024295 and WO 2005/120437 (Brunetta, P.); US 2006/0051345 and WO 2005/117978 (Frohna, P.); US 2006/0062787 and WO 2006/012508 (Hitraya, E.); US 2006/0067930 and WO 2006/31370 (Lowman et al.); WO 2006/29224 (Ashkenazi, A.); US 2006/0110387 and WO 2006/41680 (Brunetta, P.); US 2006/0134111 and WO 2006/066086 (Agarwal, S.); WO 2006/069403 (Ernst and Yansura); US 2006/0188495 and WO 2006/076651 (Dummer, W.); WO 2006/084264 (Lowman, H.); WO 2006/093923 (Quan and Sewell); WO 2006/106959 (Numazaki et al.); WO 2006/126069 (Morawala); WO 2006/130458 (Gazit-Bornstein et al.); US 2006/0275284 (Hanna, G.); US 2007/0014785 (Golay et al.); US 2007/0014720 (Gazit-Bornstein et al.); and US 2007/0020259 (Hansen et al.); US 2007/0020265 (Goldenberg and Hansen); US 2007/0014797 (Hitraya); US 2007/0224189 (Lazar et al.); and WO 2008/003319 (Parren and Baadsgaard).
Some scientific publications concerning treatment with anti-CD20 antibodies include: Perotta and Abuel, “Response of chronic relapsing ITP of 10 years duration to rituximab” Abstract #3360 Blood, 10(1)(part 1-2):88B (1998); Perotta et al., “Rituxan in the treatment of chronic idiopathic thrombocytopaenic purpura (ITP)”, Blood, 94:49 (abstract) (1999); Matthews, R., “Medical Heretics” New Scientist, (7 Apr., 2001); Leandro et al., “Clinical outcome in 22 patients with rheumatoid arthritis treated with B lymphocyte depletion” Ann Rheum Dis., supra; Leandro et al., “Lymphocyte depletion in rheumatoid arthritis: early evidence for safety, efficacy and dose response” Arthritis and Rheumatism, 44(9):S370 (2001); Leandro et al., “An open study of B lymphocyte depletion in systemic lupus erythematosus” Arthritis and Rheumatism, 46:2673-2677 (2002), wherein during a two-week period, each patient received two 500 mg infusions of antibodies of CD20, two 750 mg infusions of cyclophosphamide, and high-dose oral corticosteroids, and wherein two of the patients treated relapsed at seven and eight months, respectively, and have been retreated, although with different protocols; “Successful long-term treatment of systemic lupus erythematosus with rituximab maintenance therapy” Weide et al., Lupus, 12:779-782 (2003), wherein a patient was treated with anti-CD20 antibody (375 mg/m2×4, repeated at weekly intervals), further antibody applications were made every five to six months, and then maintenance therapy was received with antibody at 375 mg/m2 every three months, and a second patient with refractory SLE was treated with anti-CD20 antibody rituximab and was continuing to receive maintenance therapy every three months; Edwards and Cambridge, “Sustained improvement in rheumatoid arthritis following a protocol designed to deplete B lymphocytes” Rheumatology, 40:205-211 (2001); Cambridge et al., “B lymphocyte depletion in patients with rheumatoid arthritis: serial studies of immunological parameters” Arthritis Rheum., 46 (Suppl. 9): S1350 (2002); Cambridge et al., “Serologic changes following B lymphocyte depletion therapy for rheumatoid arthritis” Arthritis Rheum., 48:2146-2154 (2003); Edwards et al., “B-lymphocyte depletion therapy in rheumatoid arthritis and other autoimmune disorders” Biochem Soc. Trans., supra; Edwards et al., “Efficacy and safety of rituximab, a B-cell targeted chimeric monoclonal antibody: A randomized, placebo controlled trial in patients with rheumatoid arthritis,” Arthritis and Rheumatism, 46(9):S197 (2002); Edwards et al., “Efficacy of B-cell-targeted therapy with rituximab in patients with rheumatoid arthritis” N Engl. J. Med., 350:2572-2582 (2004); Pavelka et al., Ann. Rheum. Dis., 63:(S1):289-290 (2004); Emery et al., Arthritis Rheum. 50 (S9):5659 (2004); Levine and Pestronk, “IgM antibody-related polyneuropathies: B-cell depletion chemotherapy using Rituximab” Neurology, 52:1701-1704 (1999); Uchida et al., “The innate mononuclear phagocyte network depletes B lymphocytes through Fc receptor-dependent mechanisms during anti-CD20 antibody immunotherapy” J. Exp. Med., 199:1659-1669 (2004); Gong et al., “Importance of cellular microenvironment and circulatory dynamics in B cell immunotherapy” J. Immunol., 174:817-826 (2005); Hamaguchi et al., “The peritoneal cavity provides a protective niche for B1 and conventional B lymphocytes during anti-CD20 immunotherapy in mice” J. Immunol., 174:4389-4399 (2005); Cragg et al. “The biology of CD20 and its potential as a target for mAb therapy” Curr. Dir. Autoimmun., 8:140-174 (2005); Eisenberg, “Mechanisms of autoimmunity” Immunol. Res., 27:203-218 (2003); DeVita et al., “Efficacy of selective B cell blockade in the treatment of rheumatoid arthritis” Arthritis & Rheum, 46:2029-2033 (2002); Higashida et al. “Treatment of DMARD-refractory rheumatoid arthritis with rituximab” Annual Scientific Meeting of the American College of Rheumatology (Abstract #LB11), New Orleans, La. (October, 2002); Tuscano, “Successful treatment of infliximab-refractory rheumatoid arthritis with rituximab” Annual Scientific Meeting of the American College of Rheumatology, New Orleans, La. (October, 2002), published as Tuscano, Arthritis Rheum. 46:3420 (2002); “Pathogenic roles of B cells in human autoimmunity; insights from the clinic” Martin and Chan, Immunity, 20:517-527 (2004); Silverman and Weisman, “Rituximab therapy and autoimmune disorders, prospects for anti-B cell therapy”, Arthritis and Rheumatism, 48:1484-1492 (2003); Kazkaz and Isenberg, “Anti B cell therapy (rituximab) in the treatment of autoimmune diseases” Current Opinion in Pharmacology, 4:398-402 (2004); Virgolini and Vanda, “Rituximab in autoimmune diseases” Biomedicine & Pharmacotherapy, 58: 299-309 (2004); Klemmer et al., “Treatment of antibody mediated autoimmune disorders with an AntiCD20 monoclonal antibody Rituximab” Arthritis And Rheumatism, 48(9) (SEP):5624-5624 (2003); Kneitz et al., “Effective B cell depletion with rituximab in the treatment of autoimmune diseases” Immunobiology, 206:519-527 (2002); Arzoo et al., “Treatment of refractory antibody mediated autoimmune disorders with an anti-CD 20 monoclonal antibody (rituximab)” Annals of the Rheumatic Diseases, 61(10):922-924 (2002) Comment in Ann. Rheum. Dis. 61:863-866 (2002); “Future strategies in immunotherapy” by Lake and Dionne, in Burger's Medicinal Chemistry and Drug Discovery (John Wiley & Sons, Inc., 2003) (Chapter 2 “Antibody-Directed Immunotherapy”); Liang and Tedder, Wiley Encyclopedia of Molecular Medicine, Section: CD20 as an Immunotherapy Target (2002); Appendix 4A entitled “Monoclonal Antibodies to Human Cell Surface Antigens” by Stockinger et al., eds: Coligan et al., in Current Protocols in Immunology (John Wiley & Sons, Inc., 2003); Penichet and Morrison, “CD Antibodies/molecules: Definition; Antibody Engineering” in Wiley Encyclopedia of Molecular Medicine Section: Chimeric, Humanized and Human Antibodies (2002).
Further, see Looney, “B cells as a therapeutic target in autoimmune diseases other than rheumatoid arthritis” Rheumatology, 44 Suppl 2:ii13-ii17 (2005); Chambers and Isenberg, “Anti-B cell therapy (rituximab) in the treatment of autoimmune diseases” Lupus, 14(3):210-214 (2005); Looney et al., “B-cell depletion as a novel treatment for systemic lupus erythematosus: a phase I/II dose-escalating trial of rituximab” Arthritis Rheum., 50:2580-2589 (2004); Looney, “Treating human autoimmune disease by depleting B cells” Ann Rheum. Dis., 61:863-866 (2002); Edelbauer et al., “Rituximab in childhood systemic lupus erythematosus refractory to conventional immunosuppression Case report” Pediatr. Nephrol., 20(6): 811-813 (2005); D'Cruz and Hughes, “The treatment of lupus nephritis” BMJ, 330(7488):377-378 (2005); Looney, “B cell-targeted therapy in diseases other than rheumatoid arthritis” J. Rheumatol. Suppl., 73: 25-28-discussion 29-30 (2005); Sfikakis et al., “Remission of proliferative lupus nephritis following B cell depletion therapy is preceded by down-regulation of the T cell costimulatory molecule CD40 ligand: an open-label trial” Arthritis Rheum., 52(2):501-513 (2005); Rastetter et al., “Rituximab: expanding role in therapy for lymphomas and autoimmune diseases” Annu. Rev. Med., 55:477-503 (2004); Silverman, “Anti-CD20 therapy in systemic lupus erythematosus: a step closer to the clinic” Arthritis Rheum., 52(2):371-377 (2005), Erratum in: Arthritis Rheum. 52(4):1342 (2005); Ahn et al., “Long-term remission from life-threatening hypercoagulable state associated with lupus anticoagulant (LA) following rituximab therapy” Am. J. Hematol., 78(2): 127-129 (2005); Tahir et al., “Humanized anti-CD20 monoclonal antibody in the treatment of severe resistant systemic lupus erythematosus in a patient with antibodies against rituximab” Rheumatology, 44(4):561-562 (2005), Epub 2005, Jan. 11; Looney et al., “Treatment of SLE with anti CD20 monoclonal antibody” Curr. Dir. Autoimmun., 8:193-205 (2005); Cragg et al., “The biology of CD20 and its potential as a target for mAb therapy” Curr. Dir. Autoimmun., 8:140-174 (2005); Gottenberg et al., “Tolerance and short term efficacy of rituximab in 43 patients with systemic autoimmune diseases” Ann. Rheum. Dis., 64(6):913-920 (2005) Epub 2004 Nov. 18; Tokunaga et al., “Down-regulation of CD40 and CD80 on B cells in patients with life-threatening systemic lupus erythematosus after successful treatment with rituximab” Rheumatology 44(2): 176-182 (2005), Epub 2004 Oct. 19. See also Leandro et al., “B cell repopulation occurs mainly from naive B cells in patient with rheumatoid arthritis and systemic lupus erythematosus” Arthritis Rheum., 48 (Suppl 9): S1160 (2003).
Also see, Specks et al. “Response of Wegener's granulomatosis to anti-CD20 chimeric monoclonal antibody therapy” Arthritis & Rheumatism, 44(12):2836-2840 (2001) which disclosed use of four infusions of 375 mg/m2 of anti-CD20 antibody and high-dose glucocorticoids to treat Wegener's granulomatosis. The therapy was repeated after 11 months when the cANCA recurred, but therapy was without glucocorticoids. At eight months after the second course of anti-CD20 antibody, the patients' disease remained in complete remission. In another study remission of severe ANCA-associated vasculitis was reported, when anti-CD20 antibody was used in a dose of 375 mg/m2×4 along with oral prednisone at 1 mg/kg/day, which was reduced to 40 mg/day by week four, and to total discontinuation over the following 16 weeks. Four patients were re-treated with anti-CD20 antibody alone for recurring/rising ANCA titers. Keogh et al., Kidney Blood Press. Res., 26:293 (2003) reported that eleven patients with refractory ANCA-associated vasculitis went into remission upon treatment with four weekly 375 mg/m2 doses of anti-CD20 antibody and high-dose glucocorticoids.
Patients with refractory ANCA-associated vasculitis were administered anti-CD20 antibody along with immunosuppressive medicaments such as intravenous cyclophosphamide, mycophenolate mofetil, azathioprine, or leflunomide. Eriksson, “Short-term outcome and safety in 5 patients with ANCA-positive vasculitis treated with rituximab” Kidney and Blood Pressure Research, 26:294 (2003) (wherein five patients with ANCA-associated vasculitis were treated with anti-CD20 antibody 375 mg/m2 once a week for four weeks); Jayne et al., “B-cell depletion with rituximab for refractory vasculitis” Kidney and Blood Pressure Research, 26:294-295 (2003) (six patients with refractory vasculitis receiving four weekly infusions of anti-CD20 antibody at 375 mg/m2 with cyclophosphamide along with background immunosuppression and prednisolone experienced changes in vasculitic activity). A further report of using anti-CD20 antibody along with intravenous cyclophosphamide at 375 mg/m2 per dose in four doses for administering to patients with refractory systemic vasculitis is provided in Smith and Jayne, “A prospective, open label trial of B-cell depletion with rituximab in refractory systemic vasculitis” poster 998 (11th International Vasculitis and ANCA workshop), American Society of Nephrology, J. Am. Soc. Nephrol., 14:755A (2003). See also Eriksson, J. Internal Med., 257:540-548 (2005) regarding nine patients with ANCA-positive vasculitis who were treated with two or four weekly doses of 500 mg of anti-CD20 antibody; and Keogh et al., Arthritis and Rheumatism, 52:262-268 (2005), who reported that in 11 patients with refractory ANCA-associated vasculitis, treatment or re-treatment with four weekly 375 mg/m2 doses of anti-CD20 antibody reportedly induced remission by B-lymphocyte depletion.
As to the activity of a humanized anti-CD20 antibody, see, for example, Vugmeyster et al., “Depletion of B cells by a humanized anti-CD20 antibody PRO70769 in Macaca fascicularis,” J. Immunother., 28:212-219 (2005). For discussion of a human monoclonal antibody, see Baker et al., “Generation and characterization of LymphoStat-B, a human monoclonal antibody that antagonizes the bioactivities of B lymphocyte stimulator,” Arthritis Rheum., 48:3253-3265 (2003). The MINT trial with anti-CD20 antibody was conducted involving treating aggressive non-Hodgkin's lymphoma in younger patients. Pfreundschuh et al., Lancet Oncology, 7(5):379-391 (2006).
Antibody-cytotoxic agent conjugates (or “ACC”), also called antibody-drug conjugates (ADC), are known to be a type of immunoconjugate that consists of cytotoxic agent covalently linked to an antibody through specialized chemical linker. The use of ACCs for the local delivery of cytotoxic or cytostatic agents, i.e., drugs to kill or inhibit tumor cells in the treatment of cancer (see Syrigos and Epenetos (1999) Anticancer Research 19:605-614; Niculescu-Duvaz and Springer (1997) Adv. Drg Del. Rev. 26:151-172; U.S. Pat. No. 4,975,278) allows targeted delivery of the drug moiety to tumors, and intracellular accumulation therein, where systemic administration of these unconjugated drug agents may result in unacceptable levels of toxicity to normal cells as well as the tumor cells sought to be eliminated (Baldwin et al., (1986) Lancet pp. (Mar. 15, 1986):603-05; Thorpe, (1985) “Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review,” in Monoclonal Antibodies '84: Biological And Clinical Applications, A. Pinchera et al. (ed.s), pp. 475-506). Maximal efficacy with minimal toxicity is sought. Both polyclonal antibodies and monoclonal antibodies have sometimes been reported as being useful in this regard. (See Rowland et al., (1986) Cancer Immunol. Immunother., 21:183-87). Drugs that are known to be used in this regard include daunomycin, doxorubicin, methotrexate, and vindesine (Rowland et al., Cancer Immunol. Immunother. 21:183-87 (1986)). Toxins used in antibody-toxin conjugates include bacterial toxins such as diphtheria toxin, plant toxins such as ricin, small molecule toxins such as geldanamycin. Kerr et al (1997) Bioconjugate Chem. 8(6):781-784; Mandler et al (2000) Journal of the Nat. Cancer Inst. 92(19):1573-1581; Mandler et al (2000) Bioorganic & Med. Chem. Letters 10: 1025-1028; Mandler et al (2002) Bioconjugate Chem. 13:786-791), maytansinoids (EP 1391213; Liu et al., (1996) Proc. Natl. Acad. Sci. USA 93:8618-8623), and calicheamicin (Lode et al (1998) Cancer Res. 58:2928; Hinman et al (1993) Cancer Res. 53:3336-3342. Toxins may exert cytotoxic and/or cytostatic effects through diverse mechanisms including tubulin binding, DNA binding, or topoisomerase inhibition. Meyer, D. L. and Senter, P. D. “Recent Advances in Antibody Drug Conjugates for Cancer Therapy” in Annual Reports in Medicinal Chemistry, Vol 38 (2003) Chapter 23, 229-237. But many cytotoxic drugs tend to be inactive or less active when conjugated to large antibodies or protein receptor ligands.
Antibody-maytansinoid conjugates are composed of a monoclonal antibody that targets an antigen expressed on the surface of cells and a maytansine-derived compound (e.g., potent anti-mitotic drugs that inhibit microtubule polymerization) covalently linked to the antibody. Chari R V, Acc. Chem. Res. 2008 January; 41(1):98-107. With an appropriate linker, an antibody-maytansinoid conjugate (AMC) can be stable in vivo and significantly less toxic for cells that do not express the target antigen, thereby increasing the therapeutic index of the conjugate. Upon binding to the target antigen on the cell surface, an AMC is internalized, broken down in the lysosome, presumably by proteases, generating active maytansinoid metabolites which then bind and inhibit microtubules, thereby triggering cell cycle arrest and ultimately cell death, likely by apoptosis. Erickson et al, Cancer Res. 2006 Apr. 15; 66(8):4426-33. With maytansinoid conjugation, mAbs can improve targeted killing activity in vitro and in vivo.
Currently, numerous ACCs are being studied in clinical testing and pre-clinical development. Ideally, immunoconjugates could be easily administered to patients, similar to antibody therapies. One ACC in particular, called trastuzumab-SMCC-DM1 (T-DM1) has been reported to be effective in HER2-overexpressing metastatic breast cancer patients that have failed trastuzumab and chemotherapy, while also showing a favourable low toxicity profile. See Vogel C L, ASCO 2009 Abstract #1017.
Plasma clearance of antibody-maytansinoid conjugates, such as trastuzumab-SMCC-DM1 synthesized with the non-cleavable linker SMCC, is very slow vis-à-vis the clearance of antibody alone. US2005/0169933. This is in sharp contrast to plasma clearance of conjugates prepared with relatively labile disulfide bonds such as huC242-SPP-DM1. For example, the half-life for clearance of the SMCC conjugate is approximately 320 hours, while the half-life for the SPP conjugate is in the range of about 40 to 50 hours. However, the clearance of the antibody component for each type of conjugate is identical, suggesting that the difference in measured conjugate clearance rate could be due to the loss of maytansinoid from the antibody conjugate (i.e., in the case of the SPP-DM1 conjugate). The non-cleavable SMCC linkage has perhaps much more resistant maytansinoid-linker cleavage activities in vivo than the SPP-DM1 conjugate. Further, the decreased clearance rate for the SMCC linker conjugates, compared to SPP-DM1 conjugates, leads to a nearly 5-fold increase in overall maytansinoid exposure of the animal as measure by the area under the curve (AuC). This increased exposure could have a substantial impact on drug efficacy.
It has been reported that maytansinoid conjugates prepared with non-cleavable linkers such as SMCC show an unexpected increased tolerability in mice compared with conjugates prepared with cleavable disulfide linkers. US2005/0169933. For example, the tolerability of huC242-SMCC-DM1 and huC242-SPP-DM1 conjugates were compared in an acute toxicity test employing a single intravenous dose in CD-1 mice. The maximum tolerated dose (MTD) for the SMCC-DM1 conjugate was greater than the highest dose tested (150 mg/kg) while the MTD for the disulfide-linker conjugate SPP-DM1 was in the range of 45-90 mg/kg. At 150 mg/kg, all mice in the SMCC-DM1 treated group survived, while lethal toxicity was observed for all mice in the SPP-DM1 treated group by 96 hours post-treatment. Additionally, the non-reducible thioether-linked antibody-maytansinoid conjugate trastuzumab-SMCC-DM1 displayed 2 to 3-fold better tolerability in rats than the cleavable disulfide-linked trastuzumab-SPP-DM1. Lewis Phillips G D, Li G, Dugger D L, et al., Targeting HER2-positive breast cancer with trastuzumab-DM1, an antibody-cytotoxic drug conjugate, Cancer Res 2008; 68:9280-90. Therefore, it is possible that antibody drug conjugates prepared with non-cleavable linkers, such as SMCC, may exhibit favorable toxicity and pharmacokinetic parameters in preclinical models.
Although CD20 is known in the art, CD20 is not a favorable target for antibody-drug conjugation, since known anti-CD20 antibodies are very poorly internalized. Press O W, Cancer Res. 1989; 49:4906-12 and Vangeepuram N, Cancer 1997; 80 (Suppl.): 2425-30. Conjugates of CD20 antibodies have been studied previously but have not demonstrated significantly strong potency, especially when non-disulfide or acid stable linkers are used.
For example, it has been reported that non-cleavable SMCC-DM1 conjugates of an anti-CD20 antibody had the same efficacy as unconjugated antibody, while only a cleavable SPP-DM1 conjugate of the same antibody showed marginally improved efficacy in a Granta-519 xenograft model in SCID mice. Polson et al., Cancer Res., 69(6):2358-2364 (2009). Similarly, it has been reported that calicheamicin conjugates of anti-CD20 antibody made with an acid-stable amide linker did not show improved in vivo efficacy over rituximab in a Ramos xenograft model in nude mice. Only calicheamicin conjugates of rituximab made with an acid-labile dimethyl hydrazide Ac-But linker showed improved in vivo efficacy in this study. DiJoseph et al., Cancer Immunol. Immunotherapy, 56(7):1107-1117 (2007). In a different study, it was reported that acid labile adriamycin conjugates of an anti-CD20 antibody were only moderately effective against a Daudi xenograft model. Acid stable adriamycin conjugates of the same antibody were shown to be completely ineffective. Braslawsky et al., Cancer Immunol Immunotherapy, 33:367-74 (1991). Rituximab conjugated to monomethyl auristatin E (MMAE) via an enzyme-cleavable peptide linkage as rituximab-vcMMAE reportedly showed in vitro and in vivo efficacy against Ramos lymphoma cells. Law et al., Clin. Cancer Res., 10(23):7842-7851 (2004); Erratum in: Clin. Cancer Res., 11(10):3969 (2005).
Another reported approach at improving the ability of monoclonal antibodies to be effective in the treatment of B-cell disorders has been to conjugate a radioactive label to the antibody such that the label is localized at the antigen site. The CD20-targeted radio-immunoconjugates Bexxar® (131I-tositumomab) and Zevalin (90Y-ibritumomab tiuxetan) have been approved for relapsed or refractory non-Hodgkin's B-cell lymphoma patients, including patients refractory to rituximab. In a clinical setting, some rituximab-refractory patients responded to Bexxar®. Horning, J. Clin. Oncol. 2005; 23:712-9. When Zevalin and rituximab were compared in relapsed or refractory low-grade or follicular NHL, Zevalin treatment reportedly showed significantly better overall and complete response rates than rituximab treatment. Witzig, J Clin Oncol 2002; 20:2453-2463. While these clinical data suggest that anti-CD20 radio-immunoconjugates can be more effective than rituximab, they are not widely used because of additional toxicities and difficulty in administration associated with using radioactive compounds. Thus, there has been a need to develop effective anti-CD20 antibodies and conjugates that are easy to administer and have lower toxicity.
While even improved anti-CD20 antibody ofatumumab with better CDC activity than rituximab is effective and was approved for treating patients with refractory chronic lymphocytic leukemia, ofatumumab was not active on rituximab-refractory NHL patients. American Society of Hematology Meeting 2009, Abstract #935.
Thus, there continues to be a need for the development of improved and superior CD20 targeted therapeutic agents, including antibodies or antibody fragments that exhibit specificity, reduced toxicity, stability and enhanced physical and functional properties over known therapeutic agents. The instant invention addresses those needs.